- The fibrotic component of venous ulcers, lipodermatosclerosis or LDS, is a fundamental abnormality closely linked to the formation of ulcers and their failure to heal. The mechanisms underlying the excessive deposition of collagen in venous ulcers are unknown. Severe hypoxia is present at the ulcer site and, over the last few years, it has been shown that fibroblasts exposed to short-term hypoxia have increased clonal expansion and overall collagen synthesis, and increased mRNA levels of the a1(1) procollagen (COL1A1) gene. In large part, this stimulation is mediated by TGF-b1. Promoter analysis of the COL1A1 gene has shown that maximal stimulation in hypoxia occurs with constructs having 5' endpoints between -804 bp and -265 bp. The principal investigator has now found that hypoxic exposure of cultures over multiple passages leads to a fibroblast phenotype characterized by high levels of COL1A1 mRNA and, surprisingly, an attenuated response to stimulation by TGF-b1. Fibroblasts from venous ulcers have a similar phenotype and show decreased expression of TGF-b Type II receptors. The principal investigators hypothesis is that acute and long-term exposure to hypoxia have differential effects that, ultimately, may be responsible for increased collagen deposition and fibrosis. Acute hypoxic exposure results in increased COL1A1 transcription that is mediated, in large part, by TGF-b1. Prolonged exposure to hypoxia leads to a fibroblasts phenotype of high collagen production that is independent of TGF-b1. The principal investigator proposes to conduct the following studies: 1) Determine COL1A1 transcriptional activity and characterize the promoter regulatory regions in short-term hypoxia; 2) determine whether Smad proteins are important to stimulation of COL1A1 in hypoxic fibroblasts; 3) characterize the phenotype of fibroblasts from venous ulcers and whether they have decreased TGF-b receptor binding; 4) determine whether fibroblasts exposed to prolonged hypoxia develop a persistent phenotype of high synthetic activity that is independent of TGF-b1. The proposed studies should advance knowledge of the pathogenesis of venous ulceration and fibrosis.